JP4891079B2 - 免疫参照電極を有する免疫測定装置 - Google Patents
免疫参照電極を有する免疫測定装置 Download PDFInfo
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Description
第1固相化抗体を含み、固相化抗体、標的分析対象物質、標識抗体の間のサンドイッチ構造に基づいて第1信号を発生し、該第1信号が、前記標識抗体の非特異的結合の程度から生じる成分をもつ、電気化学センサである第1免疫センサと、
第2固相化抗体を含み、免疫参照センサとして機能し、前記非特異的結合の程度と同等か又は予測されうる限りに相関する第2信号を発生し、そして、試料中に存在するが前記標的分析対象物質ではない内因性又は外因性タンパク質と前記第2固相化抗体との間の免疫複合体を有する、電気化学センサである第2免疫センサと、
を含んで構成されることを特徴とする免疫センサシステムを提供することにある。
第1固相化抗体、標的分析対象物質、標識抗体の間のサンドイッチ構造に基づいて第1信号を発生し、該第1信号が、前記標識抗体の非特異的結合の程度から生じる成分をもつ、電気化学センサである第1免疫センサと、免疫参照センサとして機能し、前記非特異的結合の程度と同等か又は予測されうる限りに相関する第2信号を発生し、そして、第2固相化抗体と内因性又は外因性タンパク質との間の免疫複合体を有する、電気化学センサである第2免疫センサと、を含んで構成される免疫センサシステムに、前記標的分析対象物質に加えて該標的分析対象物質ではない前記内因性又外因性タンパク質を含有した試料を接触させること、
前記第1免疫センサ及び第2免疫センサを洗浄液で洗浄すること、
試料中の分析対象物質濃度に対する補正値の決定に、前記第1免疫センサからの第1信号及び前記第2免疫センサからの第2信号を使用すること、
を含むことを特徴とする方法を提供することにある。
塩試薬と、試料のイオン強度を増加させる充分な試料の処理手段と、を備え、試料が接触した場合にバフィーコート干渉を減少させる、全血試料を受ける(受信する)導管を含んで構成されることを特徴とする免疫測定装置を開示する。
イオン強度を増加させることで、試料が免疫センサに接触したときにバフィーコート干渉を減少させるように、免疫測定装置に、全血試料に対する塩試薬を添加すること、を含むことを特徴とする方法を開示する。
標的分析対象物質に対する第1固相化抗体、標的分析対象物質、標識抗体の間のサンドイッチ構造に基づいて電気化学信号を発生する、血液試料の免疫センサを含んで構成され、
前記免疫センサのセンサ表面が、
標的分析対象物質ではないが試料に存在する内因性又外因性タンパク質との間に免疫複合体を生成する、前記センサ表面の少なくとも一部に被覆される第2固相化抗体を含んで構成されることを特徴とする免疫センサシステムを開示する。
免疫センサ及びバルク伝導度センサを備え、血液試料を受ける(受信する)導管と、
前記センサからの信号を処理し、等価血漿分析対象物質濃度を決定する計算手段と、
を含んで構成されることを特徴とする電気化学免疫測定装置を開示する。
(a)血液試料を免疫センサに接触させること、
(b)前記血液試料をバルク伝導度センサに接触させて、その抵抗を測定すること、
(c)既知の伝導度を有し、及び、免疫センサに結合する分析対象物質の量に関連して検出可能な生成物を生成する充分な試薬を含む水溶液を、前記免疫センサ及び伝導度センサに接触させること、
(d)前記免疫センサにおいて前記生成物が生じる信号を測定すること、
(e)前記免疫センサからの信号をアルゴリズム手段によって分析対象物質濃度に変換すること、
(f)前記測定された血液試料の抵抗から血液試料のヘマトクリット値を計算すること、
(g)前記計算した分析対象物質濃度を前記血液のヘマトクリット値を用いて補正すること、
を含むことを特徴とする方法を開示する。
1.C無、R無:血液の引き込みは認められなかった。
2.C無、R有:保持チャンバを試薬で確実に被覆しなかったため結果は上記1に同じ。
3.C有、R無:数週間の間は、迅速な血液の引き込みが確認されたが、時間の経過と
共に引き込み率が低下。
4.C有、R有:血液の引き込みは迅速良好で、更に、6ヵ月(一般的な商品寿命)の
間、引き込みが持続。
表面−第1抗体(Ab1)〜分析対象物質〜Ab2−酵素 酵素+S→P 式(1)
表面〜Ab2−酵素 酵素+S→P 式(2)
表面〜分析対象物質−Ab2−酵素 酵素+S→P 式(3)
補正信号=IS−IRS 式(4)
iNet = ParamAct-ParamRef-c0(ナノアンペア) 式(5)
ここで、係数c0は、全血や血漿が未導入である場合のamp0及びampl間におけるバイアス、即ち、分析対象物質が存在しない場合のあらゆるバイアスとして決定される、カートリッジの製造ロットに固有な任意の値である。
iTCorr = iNet *(1+ c1 *(ATemp-TempC)) 式(6)
ここで、係数c1(per degree)の値は、一般に、カートリッジの製造ロットに固有のものではなく、カートリッジの製造工程に固有のものである。また、係数c1は、一般的に、相対的に小さな値となる(例えば、1〜3%)。当業者であれば、これを温度−依存実験から測定できることを認識可能である。
fCond = c2 * ResHct2 + c3 * ResHct + c4 式(7)
ここで、ResHctは、試料が試薬によって補正された後のヘマトクリット(伝導率)センサにおける抵抗である。
ResHct < MaxPlasmaCondであれば fCond = 1 に設定 式(8)
ResHct > MaxPlasmaCond 且つ fCond < MinfCondであれば、
fCond = MinfCond に設定 式(9)
ここで、MaxPlasmaCond = 1050、及びMinfCond = 0.8である。
iCorr= iTCorr/fCond 式(10)
[cTnI](ng/mL) = c6*iCorr/(c5*c6-iCorr) 式(11)
[cTnI](ng/mL) = c7*iCorr2 + c7*c8*iCorr 式(12)
[cTnI](ng/mL) = c6*iCorr/(c5*c6-iCorr)+ c7*iCorr2 + c7*c8*iCorr
= c6*iCorr/(c5*c6-iCorr)+ c7 * Corr2 + Linear* iCorr 式(13)
iCorr > 0.9*c5*c6であれば、iCorr = 0. 9*c5*c6に設定 式(14)
H2N−C6H4−OH → HN=C6H4=O+2H++2e−
1) 25〜50uLの試料が、試料注入口167に導入され、該試料注入口167からキャピラリーストップ151まで充填される。キャピラリーストップ151は、カバー及びベースの各構成要素をつなぎ合わせる接着テープに設けられた0.012インチのレーザ切除穴である。ユーザは、スナップ・フラップ(snap flap)上に取り付けられたラテックスゴムディスクを回転させて試料注入口167を閉じ、カートリッジを分析装置に挿入する。
2) 分析装置がカートリッジに接触すると、モータ駆動プランジャが箔袋161を押圧し、洗浄/分析液体が中央導管158に流れ込む。
3) 別個のモータ駆動プランジャが試料ダイヤフラム156に接触し、該試料ダイヤフラム156が、計量された試料セグメントを試料導管に沿って(試薬領域R1〜R2にかけて)押し出す。これにより、試料は、伝導度センサを介してセンサチップ153で検出される。このセンサチップはキャプチャー領域R3に配置されている。
4) 試料は、センサとの結合を促進するために制御時間だけ、予め定められ及び制御された方法下において、R2〜R5との間を試料ダイヤフラム156によって往復させられる。
5) 試料は、カートリッジの廃棄領域(R8)方向に押し出され、セルロース又は類似の吸収ウィック(wick)によって提供される受動的ポンプ157に接触させられる。このウィックを濡らす動作は、空気の流れを密閉する。この密閉によって、試料ダイヤフラム156が発生する超過圧力を排気するための排気機能が取り除かれる。能動的ベントは、図16に示した「制御エアーベント」になる。
6) 試料導管の急速な排気(モータ駆動プランジャを試料ダイヤフラム156から引き戻すことにより生じる)は、(ベントからの)空気と第2導管からの洗浄/分析液体との混合物を、図16のR5とR4との間に設けられた注入口に移動させる。試料導管において、急速な排気を繰り返すと、空気で区分けされた連続する液体セグメントが生み出され、センサチップを横断し試料注入口方向へと(即ち、R4、R3、R2、R1へと順番に)導かれる。これによりセンサは、洗浄されて余分な試薬が除去され、さらに、分析に適した試薬で湿らされるようになる。また、箔袋からの洗浄/分析液体は、中央の洗浄/分析液体導管内のR7及びR6における加えられた試薬によって、さらに補正される。
7) 洗浄/分析液体セグメントは、センサチップに対して分析液体の薄層だけが提供されるように、低速で試料注入口方向に引っ張られる。この段階で電気化学分析が実行される。分析方法は、好ましくはアンペロメトリであるが、ポテンショメトリ又はインピーダンス検出であってもよい。
8) カートリッジを分析装置から外すことができるように、分析装置の機構が後退する。
Claims (26)
- 干渉を減少させる免疫センサシステムであって、
第1固相化抗体を含み、固相化抗体、標的分析対象物質、標識抗体の間のサンドイッチ構造に基づいて第1信号を発生し、該第1信号が、前記標識抗体の非特異的結合の程度から生じる成分をもつ、電気化学センサである第1免疫センサと、
第2固相化抗体を含み、免疫参照センサとして機能し、前記非特異的結合の程度と同等か又は予測されうる限りに相関する第2信号を発生し、そして、試料中に存在するが前記標的分析対象物質ではない内因性又は外因性タンパク質と前記第2固相化抗体との間の免疫複合体を有する、電気化学センサである第2免疫センサと、
を含んで構成されることを特徴とする免疫センサシステム。 - 前記第1免疫センサ及び前記第2免疫センサが、アンペロメトリック電気化学センサであることを特徴とする請求項1に記載の免疫センサシステム。
- 前記第1免疫センサ及び前記第2免疫センサが、ポテンショメトリックセンサ、電界効果トランジスタセンサ、電気伝導度センサ、温度測定センサ、音波測定センサの群から選択されることを特徴とする請求項1に記載の免疫センサシステム。
- 当該免疫センサシステムが、試料中の分析対象物質を測定する使い捨てカートリッジであることを特徴とする請求項1〜3のいずれかに記載の免疫センサシステム。
- 前記標的分析対象物質が、血液試料中における、トロポニンI、トロポニンT、クレアチンキナーゼMB、プロカルシトニン、ヒト絨毛性性腺刺激ホルモン(HCG)、N末端プロ脳性ナトリウム利尿ペプチド(NTproBNP)、プロ脳性ナトリウム利尿ペプチド(proBNP)、脳性ナトリウム利尿ペプチド(BNP)、ミオグロビンからなる群から選択されることを特徴とする請求項1〜4のいずれかに記載の免疫センサシステム。
- 前記第2免疫センサの固相化抗体が、血漿タンパク質に対するものであることを特徴とする請求項1〜5のいずれかに記載の免疫センサシステム。
- 前記第2免疫センサの固相化抗体が、ヒト血清アルブミン(HSA)、ウシ血清アルブミン(BSA)、フィブリノゲン及びIgG fc領域からなる群から選択されたタンパク質に対するものであることを特徴とする請求項1〜5のいずれかに記載の免疫センサシステム。
- 前記試料中の内因性又は外因性タンパク質が、前記免疫センサシステムに試料を100秒間接触させている間に、前記第2免疫センサ上の固相化抗体の50%以上と結合するのに充分な濃度であることを特徴とする請求項1〜7のいずれかに記載の免疫センサシステム。
- 前記第2免疫センサの固相化抗体が、略1×10−7M〜1×10−15Mの親和定数を有することを特徴とする請求項1〜8のいずれかに記載の免疫センサシステム。
- 前記第1免疫センサ及び前記第2免疫センサの双方の抗体が、0.01〜5.0μmの直径を有する微粒子に固定されていることを特徴とする請求項1〜9のいずれかに記載の免疫センサシステム。
- 前記内因性又は外因性タンパク質が、前記第2免疫センサにおける抗体の親和定数と比較して少なくとも3桁以上の濃度で血液試料に存在することを特徴とする請求項1〜10のいずれかに記載の免疫センサシステム。
- 免疫センサシステムにおける干渉を減少させて標的分析対象物質を分析する方法であって、
第1固相化抗体、標的分析対象物質、標識抗体の間のサンドイッチ構造に基づいて第1信号を発生し、該第1信号が、前記標識抗体の非特異的結合の程度から生じる成分をもつ、電気化学センサである第1免疫センサと、免疫参照センサとして機能し、前記非特異的結合の程度と同等か又は予測されうる限りに相関する第2信号を発生し、そして、第2固相化抗体と内因性又は外因性タンパク質との間の免疫複合体を有する、電気化学センサである第2免疫センサと、を含んで構成される免疫センサシステムに、前記標的分析対象物質に加えて該標的分析対象物質ではない前記内因性又外因性タンパク質を含有した試料を接触させること、
前記第1免疫センサ及び前記第2免疫センサを洗浄液で洗浄すること、
試料中の分析対象物質濃度に対する補正値の決定に、前記第1免疫センサからの第1信号及び前記第2免疫センサからの第2信号を使用すること、
を含むことを特徴とする方法。 - 前記第1免疫センサ及び前記第2免疫センサが、アンペロメトリック電気化学センサであることを特徴とする請求項12に記載の方法。
- 前記第1免疫センサ及び前記第2免疫センサが、ポテンショメトリックセンサ、電界効果トランジスタセンサ、電気伝導度センサ、温度測定センサ、音波測定センサの群から選択されたものであることを特徴とする請求項12に記載の方法。
- 前記免疫センサシステムが、試料中の分析対象物質を測定する使い捨てカートリッジであることを特徴とする請求項12〜14のいずれかに記載の方法。
- 前記標的分析対象物質が、トロポニンI、トロポニンT、クレアチンキナーゼMB、プロカルシトニン、ヒト絨毛性性腺刺激ホルモン(HCG)、N末端プロ脳性ナトリウム利尿ペプチド(NTproBNP)、プロ脳性ナトリウム利尿ペプチド(proBNP)、脳性ナトリウム利尿ペプチド(BNP)、ミオグロビンからなる群から選択されることを特徴とする請求項12〜15のいずれかに記載の方法。
- 前記第2免疫センサの固相化抗体が、血漿タンパク質に対するものであることを特徴とする請求項12〜16のいずれかに記載の方法。
- 前記第2免疫センサの固相化抗体が、ヒト血清アルブミン(HSA)、ウシ血清アルブミン(BSA)、フィブリノゲン及びIgG fc領域からなる群から選択されたタンパク質に対するものであることを特徴とする請求項12〜16のいずれかに記載の方法。
- 前記試料中の内因性又は外因性タンパク質が、前記免疫センサシステムに試料を100秒間接触させている間に、前記第2免疫センサ上の固相化抗体の50%以上と結合するのに充分な濃度であることを特徴とする請求項12〜18のいずれかに記載の方法。
- 前記第2免疫センサの固相化抗体が、略1×10−7M〜1×10−15Mの親和定数を有することを特徴とする請求項12〜19のいずれかに記載の方法。
- 前記内因性タンパク質が、略100ng/ml以上の濃度のHSAであることを特徴とする請求項12〜20のいずれかに記載の方法。
- 前記内因性又は外因性タンパク質が、前記第2免疫センサにおける抗体の親和定数と比較して少なくとも3桁以上の濃度で血液試料に存在することを特徴とする請求項12〜21のいずれかに記載の方法。
- 前記第1免疫センサ及び前記第2免疫センサの双方の抗体が、0.01〜5.0μmの直径を有する微粒子に固定されていることを特徴とする請求項12〜22のいずれかに記載の方法。
- 前記微粒子が、カルボン酸塩修飾ポリスチレンビーズであることを特徴とする請求項23に記載の方法。
- 前記第1免疫センサ及び前記第2免疫センサが、洗浄効率のモニタに用いられることを特徴とする請求項12〜24のいずれかに記載の方法。
- 前記第1免疫センサ及び前記第2免疫センサからの信号が、不適当な抗凝固処理試料を含む異常状態にある試料の検出に用いられることを特徴とする請求項12〜24のいずれかに記載の方法。
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